Breast Cancer is one of the leading causes of death in women in the developed world [1]. High incidence triple-negative breast cancers (ER− and PR−, HER2-non amplified) present unsatisfactory treatments. In addition, ER+ breast cancers also became resistant to hormone-therapy. Thus novel treatments for breast cancer are urgently needed. Tumor progression has been recognized as the product of evolving cross-talk between tumor cells and the surrounding supportive tissue, known as the tumor stroma [2]. Cancer cells interact dynamically with several normal cell types within the extra-cellular matrix, such as fibroblasts, infiltrating immune cells, endothelial cells and adipocytes. Stromal and tumor cells exchange enzymes, growth factors and cytokines that modify the local extracellular matrix, stimulate migration and invasion, and promote the proliferation and survival of stromal and tumor cells. In the last decade, it has become increasingly evident that tumor cells create a peri-cellular microenvironment where molecules such as metalloproteinases, serine proteases, cysteine and aspartic cathepsins interact to form a pro-tumorigenic proteolytic network [3]. Extracellular proteases are thus primary targets for drug discovery because of their differential expression in cancer [4-6]
The lysosomal aspartic protease cathepsin D (Cath-D) is one of the most abundant lysosomal endo-proteinases implicated in protein catabolism. Human Cath-D is synthesized as a 52-kDa precursor that is converted to an active 48-kDa single-chain intermediate within the endosomes, and then to the fully active mature protease, that consists of a 34-kDa heavy chain and a 14-kDa light chain, in the lysosomes. Cath-D catalytic site has two critical aspartate residues (amino acids 33 and 231). Cath-D requires an acidic pH to be proteolytically active. Cath-D is massively overproduced and secreted by many solid tumors solid tumors: breast cancer, melanoma, ovarian cancer, lung cancer, liver cancer, pancreatic cancer, endometrial cancer, head and neck cancer, bladder cancer, malignant glioma [7]. Cath-D is a well-established independent marker of poor prognosis for breast cancer associated with metastasis [8, 9]. Several groups have shown that Cath-D affects both the cancer and stromal cell behaviors. The inhibition of Cath-D expression in breast cancer cells (BCC) decreases tumor growth and metastasis [10, 11]. Human pro-Cath-D cDNA transfected in cancer cells promotes cancer cell proliferation, tumor growth and angiogenesis, and metastasis [12-15]. Cath-D−/−MEF fibroblasts transfected with human pro-Cath-D cDNA produce more outgrowth in three-dimensional matrices [16]. We and others have shown that the overproduction of Cath-D by breast cancer cells leads to the autocrine specific hypersecretion of the 52-kDa pro-Cath-D into the extracellular environment [17, 18]. Pro-Cath-D is also secreted by macrophages infiltrating inflammatory tumors and by endothelial cells in response to inflammatory cytokines [19, 20]. Secreted human pro-Cath-D stimulates BCC proliferation [17, 18], fibroblast outgrowth [16], and endothelial cell growth [21]. Purified 52-kDa pro-Cath-D undergoes acid-dependent auto-activation in vitro, to form a catalytically-active 51-kDa pseudo-Cath-D, that retains 18 residues (27-44) of the pro-segment [22]. Since the extracellular microenvironment of hypoxic and inflammatory tumors is acidic due to the production of excess cellular acids [23-25], secreted 52 kDa pro-cath-D may auto-activate locally into proteolytically-active 51-kDa pseudo-Cath-D. At the low pH (6.8-5.5) found in tumors, Cath-D secreted by BCC degrades cystatin C, one of the most potent extracellular inhibitor of cysteine cathepsins [26]. This in turn enhances cysteine cathepsin proteolytic activity, revealing a new link in the protease web [27]. In addition, secreted Cath-D also affects BCC and stromal cells of the tumor microenvironment independently of its catalytic activity [13, 16]. Vetvicka's group described that Cath-D autocrine mitogenic growth factor activity on BCC is mediated by its activation peptide localized in a nine amino acid stretch (aa 36-44) within the Cath-D pro-peptide interacting with an unknown cell surface receptor [28]. Secreted also Cath-D promotes mammary fibroblast outgrowth via binding to LRP1 receptor (LDL receptor-related protein-1) [29, 30]. Collectively these findings provide good evidences of the oncogenic role of secreted pro-Cath-D by both proteolytic and non-proteolytic molecular mechanisms. Moreover, anti-Cath-D auto-antibodies [31] have been detected in the early stages of breast, melanoma, ovarian and lung cancers [32-35], indicating that Cath-D can be considered as a tumor-associated antigen (TAA).
Targeting Cath-D released in the tumor microenvironment will require the use of inhibitors of its catalytic activity but also the development of new tools inhibiting its interacting functions. The antibody-based delivery of therapeutic agents to the tumor site is an emerging field of modern anti-cancer research, which promises to concentrate bioactive molecules onto neoplastic lesions while sparing normal tissues. Originally monoclonal antibodies specific to membrane antigens on cancer cells have been used for tumor targeting applications. Alternative targets such as antibody-based targeting of proteases, which are hypersecreted in the tumor microenvironment, represent an additional attractive avenue for pharmaco-delivery applications [36].
New treatments are required for triple-negative (ER− and PR−, HER2-non amplified) and hormono-resistant breast cancers. The aspartic protease cathepsin D (Cath-D), an independent marker of poor prognosis in breast cancer, is over-expressed and hyper-secreted within the breast tumor micro-environment. Secreted Cath-D can affect the breast tumor microenvironment by degrading cystatin C, the most potent cysteine cathepsin inhibitor, and by triggering mammary fibroblast outgrowth via the LDL receptor-related protein-1, LRP1. Targeting secreted Cath-D in breast cancer thus requires the use of inhibitors of its catalytic activity and of its interacting functions.